Many human diseases are polymicrobial in nature and therefore require thinking beyond the traditional one organism-one disease concept to find solutions to combating them. Of fundamental importance is the need for an understanding of how these communities evolve from a healthy to a diseased state at a community, species and gene level. Although many model species found associated with healthy or disease conditions have been characterized in vitro, it is difficult to determine if their physiology in pure cultures are maintained in vivo in the presence of a mixed microbial community. In addition, most host-associated microbes remain uncultured (estimated at ~ 50% of those identified) therefore little is known about the uncultured species except for their 16S rDNA sequence. Understanding the physiology of uncultured species has become a major limiting step in studying the ecology of these polymicrobial infections. With the advent of new approaches in high throughput sequencing, single cell genomics as well as new tools in microbial ecology, we can now achieve a deeper and more detailed understanding of the physiological and ecological principals that govern the behavior of host-associated microbial communities. In doing so, we can also reveal the functions and biological role of currently uncultured members. Specifically in this application we will investigate oral microbial communities and seek to;1) identify the active species, that are highly correlated with high and low pH microbial processes through Stable Isotope Probing (SIP);2) identify the genes and dominant pathway(s) that are expressed and also shared between these active species through metatranscriptomics;3) investigate the spatial patterns and relationships of uncultured species with the other community members using Laser Capture Microdissection and 4) sequence the genomes of key suspected pathogens that are currently uncultured, through whole genome amplification from captured single cells. The success of this study would greatly expand our knowledge of oral microbial pathogenesis as it will help us to understand the virulence properties of uncultured species as well as known pathogens, not only in pure culture, but also in multi-species dental biofilms. We are particularly focused on revealing more about how these currently uncultivated bacteria may contribute to heath and disease processes. The present lack of such information presents a major barrier to understanding polymicrobial diseases. This will have a great impact on the future clinical management of dental caries and provide a comprehensive approach for characterizing the function of species and their interactions for other host-associated communities. PUBLIC HEALTH RELEVANCE: Many widespread human diseases, such as tooth decay (caries), result from the interactions of complex communities of microorganisms (polymicrobial diseases). This complexity severely complicates treatment strategies. Caries, for example, remains a major health issue in the United States and worldwide with a prevalence of more than 50% in young children, increasing to about 85% in the adult population. The majority of bacteria associated with polymicrobial diseases have not been well studied due to the fact that they cannot be grown in the laboratory. We propose to use new analytical methods to investigate the biological functions of these uncultured bacteria and their roles in human health. These approaches include generating the genomes and monitoring the physiology of these species in their natural environment. This new approach will provide a deeper understanding of disease progression and allow for the subsequent development of novel therapeutic approaches to battle these diseases.